LINE-1 (Long Interspersed Elements, L1) elements are the largest family of human retrotransposons, which are mobile genetic elements spreading in the human genome via RNA intermediates that are reverse transcribed in cDNA copies inserted into the genome. The L1 family of retrotransposons consists of about 0.5×106 copies which collectively account for ˜17% of the human genome (International Human Genome Consortium, 2001). Each functional L1 copy contains two open reading frames, ORF1 and ORF2, that are expressed as a bicistronic RNA. ORF1 and ORF2 encode a 40-kDa RNA-binding protein (ORF1p) and a 150 kDa polyprotein (ORF2p), respectively. ORF2p includes an N-terminal endonuclease domain and an adjacent reverse transcriptase (RT) domain (Mathias et al., 1991). Therefore, RT is expressed as part of the L1-ORF2 polyprotein. Only 80-100 L1 copies in the human genome are full-length and retrotransposition-competent (Brouha et al., 2003), whereas the vast majority of genomic L1 copies are truncated at their 5′ end and thus not mobile (Szak et al., 2002). This implies that, although not retrotransposition-competent, L1 elements have a high potential for producing RT (encoded by the central region of an intact ORF2) and highlights the notion that the transcriptional capability of the high number of genomic elements provides cells with a large source of RT activity. Notably, L1-encoded endogenous RT is generally expressed at higher levels in these cells that are characterized by a low differentiation states and high proliferation levels (e.g. transformed cells) (reviewed by Sinibaldi-Vallebona et al., 2011), while differentiated, quiescent cells offer less permissive contexts for RT expression (Shi et al., 2007). Clarification of the role of L1 RT in the tumorigenic process, if any, is needed.
To assess the role of L1 RT in cancer, two sets of experiments were carried out: in the first set of experiments, L1 RT was pharmacologically inhibited in tumorigenic cell lines using non-nucleoside RT inhibitors currently used in AIDS therapies, i.e. nevirapine or efavirenz (WO03055493A1; Mangiacasale et al., 2003; Landriscina et al., 2005; Sciamanna et al., 2005; Sciamanna et al., 2013), while in the second set, the expression of RT encoding L1 elements, the major source of RT activity in human cancer cells (Brouha et al., 2003), was downregulated by RNA interference (RNAi) (Sciamanna et al., 2005; Oricchio et al., 2007). Both approaches consistently resulted in reduced proliferation and enhanced differentiation of cancer cells, with clear changes of cell morphology and global alteration of the transcription profiles of both coding and non-coding RNAs while, by contrast, no appreciable effects were observed in non-transformed cells (Sciamanna et al., 2013). Moreover, RNAi-mediated L1 downregulation drastically reduced the tumorigenic potential of tumor cells in xenografted nude mice models of cancer (Oricchio et al., 2007). RT inhibitors exert a powerful anti-cancer effect also in in vivo assays, as efavirenz treatment of mice xenografted with a variety of human tumorigenic cells caused the arrest, or a significant reduction, of tumor progression (Sciamanna et al., 2005). In the last few years these conclusions were confirmed by the results from other laboratories which exposed various human tumorigenic cell lines to both nucleoside (Rossi et al., 2009; Carlini et al., 2010; Aschacher et al., 2012) and non-nucleoside (Stefanidis et al., 2008; Dong et al., 2013; Patnala et al., 2014) RT inhibitors. On these grounds, L1 RT has been regarded as a potential target in phase II trials to assess the efficacy of a novel cancer differentiation therapy (Houédé et al., 2014). Since L1-encoded ORF1p and ORF2p are detected in a variety of human cancer tissues (Rodic et al., 2014; Harris et al., 2010; Chen et al., 2012; Wang et al., 2013; Su et al., 2007), L1-ORF2 encoded RT is an attractive target for new anticancer therapies.
The time at which L1 RT becomes expressed during cancer onset or progression and the variations in L1 RT expression level during cancer progression are less clear. In a murine model of spontaneous breast cancer (MMTV-PyVT transgenic mice), Gualtieri et al., 2013 found, using a polyclonal antibody directed against mouse LINE-1 ORF2p, that L1-ORF2p is not expressed in healthy tissue but is expressed already at the first stage of cancerous transformation, and that its global (cytoplasmic+nuclear) expression increases with cancer progression.
However, Chen et al., 2012 found, using an affinity purified rabbit polyclonal anti-L1-ORF2p, that L1-ORF2p is not expressed in human non-tumor cell lines or tissue, but is expressed in breast ductal carcinomas in situ (DCIS) and in invasive breast cancer, but that global (cytoplasmic+nuclear) L1-ORF2p expression is higher in breast DCIS than in invasive breast cancer, and thus decreases with breast cancer progression.
In addition, Rodic et al., 2014 found that the level of LINE-1 expression was correlated to histological hallmarks of aggressive neoplasms, and suggest that “LINE-1 expression may be an acquired feature not seen frequently in early pre-neoplastic lesions or low-grade tumors, but rather restricted to high-grade lesions at more advanced phases of tumorigenesis”, which is also in contrast to what has been observed in a murine model by Gualtieri et al., 2013.
The reasons for the discrepancy of these results are not clear. It may illustrate that findings obtained in a murine model are not predictive of corresponding findings in humans. However, it might also be linked to some unreliability of results obtained using polyclonal anti-L1-ORF2p antibodies, the high specificity of which for L1-ORF2p may be questioned. Similarly, it has to be noted that initial reports of the presence of L1 RT in cancer cells in Mangiacasale et al., 2003 were based on the use of an anti-HIV RT monoclonal antibody, the ability to specifically recognize L1-ORF2p of which may be questioned. Discrepancies may also reflect differences between different tumors or individual variability, thus making interpretation of the above mentioned results quite difficult.
WO2014004945 suggest the use of the expression level of L1 ORF1p in human patients, in particular in their serum or plasma, as a biomarker of cancer, for early diagnosis of cancer. However, ORF1 and ORF2 are two different coding domains of L1 elements, exhibiting different localization, structure and biological functions: the former is an RNA-binding protein while the latter codes for reverse transcriptase and nuclease enzymes. Study of ORF1 could in no way suggest predictions for the role(s) of ORF2. This further illustrated by the fact that no connection between the levels of ORF1p and ORF2p expression was found in another study (Dai et al., 2014).
In addition to the importance of determining the potential causative role of L1 RT in cancer onset and progression, it is also crucial to be able to detect, for a particular cancer patient, whether or not its cancer cells express human L1-ORF2p. Indeed, as explained above, human L1-ORF2p is believed to be expressed in a number of cancers, and non-nucleoside reverse transcriptase inhibitors (NNRTI) and in particular efavirenz, have been proposed as new anticancer treatments (see Mangiacasale et al., 2003; Landriscina et al., 2005; Sciamanna et al., 2005; Sciamanna et al., 2013). However, administering NNRTI treatment to a subject whose cancer cells do not express human L1-ORF2p would be useless, and the ability to reliably detect human L1-ORF2p in cancer tissue, with high sensitivity and specificity is thus of huge importance.
There was thus a need for a reliable mean to measure L1-ORF2p expression, in order to be able to reliably assess the expression of L1-ORF2p in precancerous lesions and in various stages of cancer.
However, since L1-ORF2p amino acid sequence is well conserved in mammals, and more generally in vertebrates, the production of an anti-L1-ORF2p antibody was not an easy task, since human L1-ORF2p is very close to non-foreign animal L1-ORF2p. In particular, human and mouse L1-ORF2p amino acid sequences are extremely well conserved and differ from each other only in few regions of the amino acid sequence. A mouse will normally generate antibody responses only against protein or peptides recognized as foreign. In addition, it is well known in the art that not all peptides of a protein are immunogenic. As a result, generating a murine anti-human L1-ORF2p monoclonal antibody was not obvious.
In some cases, antibodies directed to other reverse transcriptases (RT), such as HIV RT, have been used for detection of human L1-ORF2p (Mangiacasale et al., 2003). However, due to differences between HIV RT and human L1-ORF2p, recognition of human L1-ORF2p is not really specific and not very sensitive (see Example 5 below).
In addition, despite the above mentioned difficulties, several anti-human L1-ORF2p antibodies have nevertheless been generated. However, these antibodies are not suitable for the above mentioned purpose of reliably detecting human L1-ORF2p in cancer tissue, with high sensitivity and specificity. In particular, prior art anti-human L1-ORF2p antibodies were generally polyclonal, which means that they are available only until all generated polyclonal antibody has been used, which is not appropriate for clinical detection, for which durable use of the same reference anti-human L1-ORF2p antibody is desirable. Moreover, while prior art anti-human L1-ORF2p antibodies are able to detect overexpressed human L1-ORF2p when cells are transfected by an expression vector of human L1-ORF2p, they are generally unable to detect endogenous human L1-ORF2p naturally expressed by cancer cells (see Examples 5 and 7 below). Furthermore, while tumor tissues are generally available as tissue sections and a preferred mean for detecting human L1-ORF2p expression would be immunohistochemistry, prior art antibodies are unable to detect endogenous human L1-ORF2p expression in cancer tissue (see Example 7 below).
Notably, Goodier et al., 2004 disclose two rabbit polyclonal anti-human L1-ORF2p antibodies: an anti-ORF2-C antibody directed to amino acids 1259-1275 of human L1-ORF2p sequence and an anti-ORF2-N polyclonal antibody directed to amino acids 154-167 of human L1-ORF2p sequence (peptide DRSTRQKVNKDTQE (SEQ ID NO:9) of the endonuclease domain). FIG. 2 of Goodier et al., 2004 presents immunoblot analysis of L1-ORF2p in an artificial system constituted by human 143B osteosarcoma cells transfected with a L1-ORF2p-overexpressing construct using the two above mentioned rabbit polyclonal anti-human L1-ORF2p antibodies. As clearly shown, only overexpressed L1-ORF2p can be detected (lanes 1-3,8), while endogenously expressed protein is not detected at all (lane 7). Moreover, when using the anti-ORF2-N polyclonal antibody directed to peptide DRSTRQKVNKDTQE (SEQ ID NO:9) of the endonuclease domain, high background is present in lanes 2-3. One of these two rabbit polyclonal anti-human L1-ORF2p antibodies has also been used in immunoblot analysis of human L1-ORF2p in an artificial system constituted by HeLa cells transfected with various human L1-ORF2p-overexpressing constructs (see FIG. 2B of Doucet et al., 2010). Clearly, endogenous human L1-ORF2p is also not detected in non-transfected HeLa cells using this antibody. In addition, no data has been provided showing that these rabbit polyclonal anti-human L1-ORF2p antibodies would be able to detect human L1-ORF2p by immunohistochemistry.
Sokolowski et al. recently disclosed a murine monoclonal antibody directed to human L1-ORF2p (Sokolowski M et al, 2014). This murine monoclonal antibody directed to human L1-ORF2p has been generated by immunizing mice with a bacterially purified endonuclease domain of the human L1-ORF2 protein. While the epitope recognized by this antibody has not been completely identified, it has been shown that amino acid 205 is included into the recognized epitope. Data presented in this article show that this antibody recognizes overexpressed but not native L1-ORF2p in HeLa cell line, and is thus not enough sensitive to detect endogenous L1-ORF2p in cancer cell lines. In addition, only immunoblot data are presented in this article, and there is thus no demonstration that this antibody is able to detect L1-ORF2p in immunochemistry assays, on cancer sample tissues.
Further attempts to generate antibodies to L1-ORF2p are disclosed in Table 1 of Dai et al., 2014. Among all tested antibodies directed to L1-ORF2p, most detected only overexpressed ORF2p (see column “Endogenous or overexpressed”) and not endogenous L1-ORF2p. Only two are mentioned to detect endogenous ORF2p, one is directed to rat L1-ORF2p and does not detect above background level human ORF2p of non-transfected HeLa cells (see FIG. 8B of Kirilyuk et al, 2008), the other is a polyclonal antibody (see Chen et al, 2012). Therefore, despite several attempts to generate monoclonal antibodies able to recognize human L1-ORF2p, none of the antibodies of the prior art was able to detect endogenous human L1-ORF2p.
However, for the above mentioned purpose of selecting cancer patients for NNRTI treatment based on expression of L1-ORF2p by their cancer, a monoclonal anti-human L1-ORF2p antibody that is both sensitive enough to detect endogenous L1-ORF2p in cancer cells and able to detect L1-ORF2p in immunochemistry assays, on cancer sample tissues, is needed.